This research project will be performed at the University of Washington as a collaboration between investigators in the Departments of Medicine, Chemistry, and Biochemistry. The group of scientists consisting of Drs. Buckner (biology), Gelb (chemistry), and Verlinde (structure-based drug design) has worked as a team on antiparasitic drug development for ~15 years with collectively >100 published articles in the field. The work will be done using state-of-the-art facilities and equipment, as well as expert research staff, ensuring a productive and successful research program. The long-term objective of the proposed research is to develop inhibitors of the sterol 14a-demethylase (14DM) enzyme for treating Chagas disease. Trypanosoma cruzi, the etiologic agent of Chagas disease, is widespread in Latin America and causes cardiomyopathy and pathological dilations of the gastrointestinal tract. The currently available treatments for this disease are inadequate because of poor efficacy and toxicity. In the previous funding period, our research showed that inhibitors of sterol biosynthesis are highly potent in vitro and in animal models of Trypanosoma cruzi infection. Specifically, a series of compounds related to a cancer drug, tipifarnib, was discovered to be highly active against a key enzyme (14DM) in the sterol biosynthesis pathway. More research is needed to identify analogs of tipifarnib that are optimized for activity against Chagas disease. In the next funding period, the group proposes to pursue the following specific aims: 1) Design and synthesize of ~100 tipifarnib analogs per year using rational design methods. A crystal structure of the T. cruzi 14DM solved in 2010 by one of our collaborators will be used to guide design of new tipifarnib analogs. Physicochemical properties and analysis of metabolites from liver microsome incubations will help direct the synthesis plans to make bioavailable compounds with good metabolic stability. 2) Test the efficacy, pharmacology, and toxicity of tipifarnib analogs. The newly synthesized compounds will be tested in a series of studies and advanced or terminated by pre-defined criteria. All compounds will be tested in binding assays on the T. cruzi 14DM enzyme, for inhibition of T. cruzi amastigotes, and for cytotoxicity against mammalian cells. Follow up studies will test aqueous solubility, stability in liver microsomes, and inhibition of human CYP450 enzymes. Compounds will next be tested for pharmacokinetic properties in rodents and the best compounds will be tested for efficacy in the mouse model of established T. cruzi infection. Selected compounds will be tested for hERG channel inhibition, human receptor profiling, and genotoxicity studies. 3) Screen for synergy partner drugs. From a screen of 2000 compounds, we have identified 60 FDA approved drugs with inhibitory activity on T. cruzi amastigotes. Eight have been shown to have synergistic activity when combined with the tipifarnib analog (JK-11). In this Aim, we will move from in vitro assays to testing the combinations in the mouse model of T. cruzi infection. We hope to identify at least one FDA approved, orally administered drug that boosts the in vivo efficacy of the lead tipifarnib analog. By identifying a partner drug, we envision the possibility of an anti-Chagas drug regimen that is shorter than current treatment courses of 60-90 days and stems development of potential drug resistance.